Co-activator independent differences in how the metaphase and anaphase APC/C recognise the same substrate.

Matsusaka T, Enquist-Newman M, Morgan DO, Pines J - Biol Open (2014)

Bottom Line:
We have addressed this question by determining whether the same substrate, cyclin B1, is recognised in the same way by the APC/C at different times in mitosis.Unexpectedly, we find that distinct but overlapping motifs in cyclin B1 are recognised by the APC/C in metaphase compared with anaphase, and this does not depend on the exchange of Cdc20 for Cdh1.Thus, changes in APC/C substrate specificity in mitosis can potentially be conferred by altering interaction sites in addition to exchanging Cdc20 for Cdh1.

f01: L45 of the Cyclin B1 D-box is most important for degradation.(A) Schematics of cyclin B1 constructs used in this study. The D-box is highlighted in blue. (B,C) HeLa cells were injected with cyclin B1-Venus (grey, n = 36) or cyclin B1 L45A-Venus (red, n = 24) constructs and followed by time-lapse fluorescence and DIC microscopy at 3-min intervals. The total fluorescence minus background was quantified for each cell in successive images of a time series and plotted over time as mean ± SD from 3 independent experiments. Fluorescence of cells at NEBD (B) or anaphase onset (C) was set to 1. Time 0 is NEBD in (B) or anaphase onset in (C). (D,E) HeLa cells were injected with cyclin B1-Venus (grey, n = 36), L45A-Venus (black, n = 24) or cyclin B1 R42A-Venus (red, n = 55) constructs and analysed as in panels B and C. Fluorescence of cells at NEBD (D) or anaphase onset (E) was set to 1. Time 0 is NEBD in (D) or anaphase onset in (E). Data are from 3 independent experiments. (F,G) HeLa cells were injected with cyclin B1-Venus (grey, n = 36), L45A-Venus (black, n = 24) or cyclin B1 N50A-Venus (red, n = 33) constructs and analysed as in panels B and C. Fluorescence of cells at NEBD (F) or anaphase onset (G) was set to 1. Time 0 is NEBD in (F) or anaphase onset in (G). Data are from 3 (wt and L45A) or 2 (N50A) independent experiments. (H,I) HeLa cells were injected with cyclin B1-Venus (grey, n = 36), L45A-Venus (black, n = 24) or cyclin B1 R42A/N50A-Venus (red, n = 38) constructs and analysed as in panels B and C. Data are from 3 independent experiments.

Mentions:
We first analysed the effect of mutating those conserved residues of the cyclin B1 D-box that had previously been shown to be most critical for destruction: R42, L45, and N50 (King et al., 1996; Yamano et al., 1998) (Fig. 1A). L45 proved to be essential for degradation in both metaphase and anaphase (Fig. 1B,C). The likely explanation for this is that the structure of the putative D-box binding site on Cdc20 indicated that L45 should be buried in a deep pocket (Chao et al., 2012). By contrast, we found mutating R42, which is commonly used to inactivate a D-box, only partially stabilised Cyclin B1 in anaphase (Fig. 1D,E). Mutating N50 had a similar effect to mutating R42 (Fig. 1F,G), but mutating both residues stabilised cyclin B1 in both metaphase and anaphase, in a similar fashion to mutating L45 (Fig. 1H,I).

f01: L45 of the Cyclin B1 D-box is most important for degradation.(A) Schematics of cyclin B1 constructs used in this study. The D-box is highlighted in blue. (B,C) HeLa cells were injected with cyclin B1-Venus (grey, n = 36) or cyclin B1 L45A-Venus (red, n = 24) constructs and followed by time-lapse fluorescence and DIC microscopy at 3-min intervals. The total fluorescence minus background was quantified for each cell in successive images of a time series and plotted over time as mean ± SD from 3 independent experiments. Fluorescence of cells at NEBD (B) or anaphase onset (C) was set to 1. Time 0 is NEBD in (B) or anaphase onset in (C). (D,E) HeLa cells were injected with cyclin B1-Venus (grey, n = 36), L45A-Venus (black, n = 24) or cyclin B1 R42A-Venus (red, n = 55) constructs and analysed as in panels B and C. Fluorescence of cells at NEBD (D) or anaphase onset (E) was set to 1. Time 0 is NEBD in (D) or anaphase onset in (E). Data are from 3 independent experiments. (F,G) HeLa cells were injected with cyclin B1-Venus (grey, n = 36), L45A-Venus (black, n = 24) or cyclin B1 N50A-Venus (red, n = 33) constructs and analysed as in panels B and C. Fluorescence of cells at NEBD (F) or anaphase onset (G) was set to 1. Time 0 is NEBD in (F) or anaphase onset in (G). Data are from 3 (wt and L45A) or 2 (N50A) independent experiments. (H,I) HeLa cells were injected with cyclin B1-Venus (grey, n = 36), L45A-Venus (black, n = 24) or cyclin B1 R42A/N50A-Venus (red, n = 38) constructs and analysed as in panels B and C. Data are from 3 independent experiments.

Mentions:
We first analysed the effect of mutating those conserved residues of the cyclin B1 D-box that had previously been shown to be most critical for destruction: R42, L45, and N50 (King et al., 1996; Yamano et al., 1998) (Fig. 1A). L45 proved to be essential for degradation in both metaphase and anaphase (Fig. 1B,C). The likely explanation for this is that the structure of the putative D-box binding site on Cdc20 indicated that L45 should be buried in a deep pocket (Chao et al., 2012). By contrast, we found mutating R42, which is commonly used to inactivate a D-box, only partially stabilised Cyclin B1 in anaphase (Fig. 1D,E). Mutating N50 had a similar effect to mutating R42 (Fig. 1F,G), but mutating both residues stabilised cyclin B1 in both metaphase and anaphase, in a similar fashion to mutating L45 (Fig. 1H,I).

Bottom Line:
We have addressed this question by determining whether the same substrate, cyclin B1, is recognised in the same way by the APC/C at different times in mitosis.Unexpectedly, we find that distinct but overlapping motifs in cyclin B1 are recognised by the APC/C in metaphase compared with anaphase, and this does not depend on the exchange of Cdc20 for Cdh1.Thus, changes in APC/C substrate specificity in mitosis can potentially be conferred by altering interaction sites in addition to exchanging Cdc20 for Cdh1.